Autophagy research in the cardiovascular system has focused almost exclusively on macroautophagy, in which double-membrane vesicles transport molecules and organelles to lysosomes for degradation. In contrast, the subject of this project is a distinct process termed chaperone mediated autophagy (CMA). In CMA, cytoplasmic proteins are selectively targeted for degradation through a mechanism in which Hsc70 and co-chaperones bind a recognition motif on the target protein. This complex then translocates to the lysosome where it is imported into the lumen by LAMP2A (L2A), a lysosomal transmembrane protein that is necessary, specific, and rate-limiting for CMA. Our informatics analyses suggest that there are ~7000 potential CMA substrates in the heart, and a significant proportion of these proteins are dynamically regulated. However, there has been no means to assess the functional significance of CMA in healthy or diseased hearts until recently, when we generated mice with an inducible, cardiomyocyte-specific knockout of LAMP2A (iCS- L2AKO). While these mice are normal at baseline, an unanticipated phenotype emerges when they are stressed with pressure overload or post-myocardial infarction heart failure: Systolic dysfunction in each of these models is attenuated by inhibition of CMA ? not worsened, as might be expected from the traditional role of autophagy in ameliorating cellular stresses. Mechanistic investigations revealed another unexpected relationship: Inhibition of CMA induces mitophagy, a process that maintains the overall health of the mitochondrial pool by eliminating defective organelles. We propose a new paradigm in which CMA, activated in response to cardiac stress, mediates cardiac dysfunction by depleting cardiomyocytes of proteins that would normally promote mitochondrial quality control through mitophagy. We will test this model and delineate molecular mechanisms that link activation of CMA with suppression of mitophagy. Aim 1, will define the functional role of CMA in heart failure, using both pressure overload and MI models. These studies will employ multiple innovative reagents including mouse models of inducible, cardiomyocyte-specific L2A deletion and overexpression, a recently developed small molecule activator of CMA, and a new CMA reporter mouse. We will also define functional relationships between CMA and macroautophagy in the heart. Aim 2 will identify molecules that mediate the suppression of mitophagy by CMA. We will investigate a role for a strong candidate: the mitophagy activator Parkin, which our studies suggest is a CMA substrate. In addition, to identify novel mediators, we propose an unbiased approach that combines lysosomal proteomics (to identify direct CMA substrates) and RNA-seq (to identify mediators regulated indirectly by CMA). The proposed experiments are highly significant and innovative in that they will provide the first assessment of the role of CMA in the heart and define a novel heart failure pathway that is mediated by previously unrecognized connections between CMA and mitophagy.